The goals of the proposed research are to develop methods for solving structures of eukaryotic membrane proteins and characterization of their complexes with other membrane proteins and small-molecule substrates. Structure determination of membrane proteins is still a challenge, and relatively few structures of integral membrane proteins have been solved. Moreover, most of these structures are from bacteria or plants, and to our knowledge, only one human integral membrane protein structure has been solved so far. Here we propose to solve the structure of the voltage-dependant anion channel, VDAC, a 300-residue integral outer membrane protein from human mitochondria. We will also determine the structure in detergent micelles of the complex of VDAC with the anti-apoptotic protein Bcl- xL and/or other proteins from the Bcl-2 family. This system is of high significance for the mitochondrial apoptosis pathway. In this project we will develop and optimize methods for expression, isotope labeling, refolding and reconstitution in membrane mimicking environments. We will develop improved methods for resonance assignments and structure determination for difficult membrane-associated systems, and we will develop procedures for defining protein-protein and protein-substrate complexes in membrane-like environments. The research will pursue the following specific aims: 1. Development and optimization of methods for expression, isotope labeling and reconstitution of VDAC in a membrane-like environment 2. Methods for resonance assignments and structure determination in a membrane-like environment 3. Structure of VDAC in a detergent micelle 4. Resonance assignments and structure determination of micelle-bound of Bcl- xL 5. Structure of the VDACl/Bcl-xL complex 6. Discover inhibitors of the interaction between VDAC and Bcl- xL -like proteins.